Applied Toxicology

NURS 735

METABOLISM

Andrew S. Kane, Ph.D. UM Program in Toxicology Director, UM Aquatic Pathobiology Center, UMCP
akane@umaryland.edu

This PowerPoint presentation will review basic principles of biotransformation relative to the metabolism of xenobiotics taken into the body. Focus will be on primary phase I and phase II pathways; types of enzymes, localization of enzymes enzyme function and interand intra-individual differences. Additional materials on different enzyme pathways and ADME examples are included in the textbook and the hyperlinked reading assignments. This ppt presentation was developed to help clarify the subject of ADME and is supplemental to your other reading assignments.

A.S. Kane, NURS 735

Xenobiotics
Accumulation
(storage in body fat, bone) highly lipophilic metabolically stable lipophilic polar hydrophilic

Phase I metabolism (bioactivation or inactivation)

oxidation, reduction, hydrolysis polar

Phase II metabolism (bioinactivation) conjugation

hydrophilic

Extracellular mobilization Plasma circulation Biliary excretion Renal excretion

The concept of metabolism (i.e., biotransformation) applies not only to xenobiotics but to endogenous chemicals as well (biotransformation is critical for the processing and recycling of endogenous compounds). The above figure illustrates the concept of xenobiotic disposition in the body. Note that compounds taken into the body may remain unchanged (and possibly accumulate in a storage compartment), or based on their degree of lipophilicity and polarity, be subject to metabolism. In general, lipophilic compounds have a tendency to pass through biological membranes and/or be stored, and are often susceptible to phase I types of metabolism. The metabolite(s) of phase I reactions tend to be more polar, thus facilitating direct excretion or phase II metabolism. Phase II types of metabolism generally involve conjugation reactions. These conjugations help to make the compounds even more polar, thus facilitating biliary and renal excretion.

A.S. Kane, NURS 735

This flow chart highlights the role of metabolism in the disposition of various compounds prior to excretion or causing pharmacologic or pathologic effects. Compounds that are metabolized may be either detoxified or activated. In general this concept of detoxification or bioactivation applied to phase I metabolism (phase II pathways tend not to activate, although there are exceptions to most rules as we will see later in this ppt presentation).

A.S. Kane, NURS 735

This table lists examples of different enzyme-mediated pathways by reaction type. In this introductory course we will focus on cytochrome P450s as the primary phase I enzyme systems and GT, ST and GST as examples of phase II enzyme systems. However, it is important to know that there are other important functional pathways. The next two slides will present information about where the enzymes are located.

A.S. Kane, NURS 735

The intracellular localization of phase I cytochrome P450 enzymes is within smooth endoplasmic reticulum (SER). The cartoon above shows how different biotransformation enzymes (both phase I & II) are integral within the lipid bilayer of the SER (some enzymes are transmembrane proteins). Note the presence of a cytochrome P450 enzyme complex, including cytochrome b5, and the presence of cofactors (NADPH is required as an electron donor). Also present are glucuronyltransferase enzymes (denoted as GT). The GT enzymes are phase II enzymes localized nearby. This intimate relationship between cyp450 and GT facilitates coupling between certain phase I and phase II reactions.

A.S. Kane, NURS 735

Localization of phase II conjugation reactions

Localization of phase II enzymes includes both SER and cytosol. This table includes a listing of endogenous substrates, reminding us that the biotransformation pathways used for detoxification are equally important in recycling many compounds withiin the body and maintaining biochemical homeostasis.

A.S. Kane, NURS 735

Phase I reactions

Derelanko, M. and Hollinger, M. CRC Handbook of Toxicology, 1995

Lets start by talking about some of the enzymes that mediate phase I reactions. Most of these enzymes are members of an enzyme superfamily known as cytochromes P450. The table above shows some of the P450 families (connoted by the number after CYP) and subfamilies (connoted by the letter after CYP+number), some known inducing agents (well get back to this later in this ppt), and characteristic reactions (i.e., hydroxylation, demethylation, etc.). Although you are not responsible for recalling all the different P450s, you should know that: (1) this is a group of enzymes, (2) different enzymes within this group have varying affinities for specific substrates (chemicals), (3) certain enzymatic reactions lead to specific types of products (metabolites), and (4) different enzyme reactions can be altered (inhibited or induced) by specific types of agents or exposures.

A.S. Kane, NURS 735

Absorbance spectrum, 400-500 nm (CO difference spectra), of cytochrome P450

How did cytochrome P450 get its name? Shown here is the carbon monoxide difference spectra for a cytochrome P450. In a reduced (Fe+2) state, this enzyme can bind to ligands such as CO. The complex between cyp P450 and CO absorbs light maximally at approximately 450 nanometers, hence its name derivation.

A.S. Kane, NURS 735

Overview of possible types of phase I biotransformation reactions

Oxidation reactions: Loss of electrons, often addition of O to replace H

Reduction reactions: Gain of electrons, often addition of H to replace O

Hydrolysis reactions: Water interacts with substrate such that O 2 makes bond

Cytochrome P450 enzymes catalyze several types of oxidation reactions including hydroxylation of an aliphatic or aromatic carbon; epoxidation of a double bond; heteroatom oxygenation, hydroxylation or dealkylation; oxidative group transfer, cleavage of esters and dehydrogenation. The broad and often overlapping substrate specificity of these enzymes makes it impossible to name the enzyme for specific reactions. Cytochrome P450 enzymes are microsomal, i.e., they are concentrated in centrifugally-derived laboratory preparations enriched in SER. Liver tissue has the highest concentration of most biotransformation enzymes, although other organsdo have varying degrees of metabolic capacity.

A.S. Kane, NURS 735

Bioactivation

We mentioned that biotransformation through phase I pathways can lead toward detoxification, either directly or via continued phase II metabolism. This slide addresses the concept of bioactivation via phase I metabolism, i.e., toxicity occurring due to metabolism. Shown here are reactive intermediates formed due to phase I activation. Although these intermediates may be further metabolized and detoxified, they can also lead to pathology.

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Bioactivation by Phase I enzymes Aflatoxin B1 Aspergillus flavus Penicillium puberculum 1ppb carcinogenic to rats in chronic exposures

Here we have an example of bioactivation via phase I metabolism. In this example, aflatoxin B1 is metabolized to form a reactive epoxide (note the loss of a double bond, replaced with an epoxide, in the middle portion of the diagram, left side of the molecule). This electrophilic epoxide can covalently bind to DNA, leading to neoplasia. Alternatively, the epoxide can be conjugated with glutathione. Both reactions can occur in the same animal. The question of whether or not cancer develops is related to the rate of glutathione conjugation, the rate of DNA binding, and the effectiveness of DNA repair mechanisms. Aflatoxin is a product of certain molds that can be found in improperly stored foodstuffs.

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Benzo[a]pyrene is a good example of a carcinogen requiring phase I activation to exert carcinogenic potential. Although you will not be required to reconstruct this flow chart, note that there are multiple pathways that can occur. The dynamics of these pathways and subsequent repair mechanisms determine the likelihood of tumorogenesis. There are two cytochrome P450 pathways that can form reactive intermediates (epoxides). The 4,5 epoxide does not have nearly the carcinogenic potential as the 7,8 epoxide. Note the the epoxides may form diols through epoxide hydrolases, and that the 7,8 dihydrodiol can be further metabolized by cytochrome P450 to yield a 7,8 dihydrodiol, 9,10 epoxide. Due to the configuration of this molecule, it can readily intercalate into DNA, leading to altered cell growth and cancer.

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Now, lets focus on phase II reactions, most of which involve conjugating a substrate to another molecule (or part of a molecule). This conjugation generally renders the product to be more polar. An increase in the polarity facilitates excretion. In order for phase II metabolism to occur, the substrate must have a functional handle on its molecule to support the conjugation reaction (a place that facilitates conjugation to a donor molecule such as glucuronic acid moiety or a sulfate moiety. For example, glucuronide or sulfate conjugation reactions may occur at the site of an hydroxyl or carboxyl group on the substrate molecule; for glutathion conjugations, epoxides or organic halides are common joining moieties. Note that phase II substrates may be parent compounds or metabolites of phase I reactions.

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Phase II Conjugation Enzyme Cofactors

We already learned that cofactors are necessary for most phase I reactions. Cofactors are also essential for phase II reactions (to provide the donor polar molecule). Examples are given above. In the case of glucuronidation, (uridine diphosphate glucuronic acid (UDPGA) is the necessary cofactor, and serves as the donor of a glucuronic acid moiety. What are the respective cofactors necessary for ST and GST reactions?

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Sulfotransferase-mediated conjugation

Here is a detailed diagram illustrating the ST-mediated conjugation of phenol. Note that the phenyl sulfate product contains only the sulfate portion of the donor molecule, not the PAP portion; this gets biochemically recycled for subsequent reactions in the body.

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Example of (indirect) bioactivation via phase II reactions:

Glucuronides may be unstable under certain circumstances (eg some aromatic amines). Compounds may be bio-activated in the liver to form N-hydoxyl derivatives, after which they can be bio-inactivated by forming N-glucuronides. However, in an acid environment (i.e., urine), the glucuride is a good leaving group, allowing the substance to exert a possible carcinogenic effect on the bladder epithelium.

Typically, we think of only phase I reactions being able to metabolically bioactivate a substrate. However, as there are exceptions to most rules, here is an example of bioactivation via a phase II pathway.

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Organ and species differences in aromatic hydroxylation of benzo[a]pyrene

There are many differences in metabolic capacity between species, some of which are indicated in the table above. As you are now well aware, there are also intra-specific differences associated with age, gender, nutrition, genetic predisposition and environmental factors including various exposures that may cause enzyme induction or inhibition.

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Example of therapy to reduce acetaminophen toxicity by fostering GST conjugation of a reactive intermediate.

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Species Differences Dogs do not acetylate aromatic amines Cats are deficient in N-actyltransferase & glucuronyltransferase Guinea pigs do not form mercapturic acid conjugates Pigs are deficient in sulfate conjugation

Inter-individual Differences Genetic differences (extensive vs poor metabolizers) Sex differences Age differences Other environmental stressors

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Pathway shift in poor vs extensive metabolizers

Primary pathway in extensive metabolizers is O-dealkylation to acetaminophen. However in poor metabolizers the deacetylation reaction is favored leading to subsequent hydroxylation and a 2-hydroxyphenetidin product. This product can bind to and oxidize hemaglobin to form methemaglobin.

Example of inter-individual differences in phase I metabolic capacity.

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Dietary Metabolic Alterations

IN MOUSE HEPATIC MICROSOMES, DIETARY BUTYLATED HYDROXYANISOLE (BHA) ENHANCED THE TOTAL METABOLISM OF BENZO(A)PYRENE (BP) BUT DECREASED THE MICROSOMAL METABOLISM OF (BP)-7,8-DIOL, ESPECIALLY THE FORMATION OF (BP)-TRANS-7,8-DIOL-ANTI-9,10-OXIDE. THE ALTERED METABOLISM OF BENZO(A)PYRENE IS BELIEVED TO BE DUE TO THE INDUCTION OF NEW CYTOCHROME P450 SPECIES BY DIETARY BHA.

THUS, BHA CAN AFFECT BENZO(A)PYRENE METABOLISM BY EXERTING ITS INHIBITORY EFFECT DIRECTLY & BY ALTERING THE COMPOSITION OF MICROSOMAL MONOOXYGENASE ENZYMES AFTER A FEW DAYS OF EXPOSURE.
[SYDOR W JR ET AL; CARCINOGENESIS 4 (2): 131-6 (1983)]

Diet is just one factor that can alter metabolic kinetics.

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Age-dependent development of glucuronidation

Another example of inter-individual differences in metabolic capacity. Line a refers to normal neonates and their glucuronysyl transferase activity. There is an increase in activity up until birth, whereupon it peaks and then levels off. Line b refers to a subset of the population where GT activity does not peak until weeks after birth. These neonates are subject to jaundice (failure to conjugate bilirubin) and require the use of bili lights. Using bili lights is a therapeutic procedure performed on newborn or premature infants to reduce elevated levels of bilirubin. If blood levels of bilirubin become too high, the bilirubin begins to dissolve in the body tissues, producing the characteristic yellow eyes and skin of jaundice. Bilirubin also has an affinity for brain tissue, where it can accumulate and cause permanent brain damage.

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Preferred route of excretion of xenobiotic conjugates

The route of excretion may vary with the type(s) of conjugation reaction favored and the molecular weight of the metabolite(s).

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Routes of Elimination
Expired Air Urine Bile Feces Saliva M ilk Hair Volatile Compounds M ajor route for low molecular weight polar compounds High molecular weight, conj ugated compounds Compoundsnot abs orbed in gut or compoundsexcreted in the bile Very low molecular weight compounds Both water and lipid s oluble compounds Quantitatively unimportant

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